Alansary, D. | Search bioRxiv

bioRxiv2020

Alternative splicing switches STIM1 targeting to specialized membrane contact sites and modifies SOCE

Alternative splicing is a potent modifier of protein function. Stromal interaction molecule 1 (Stim1) is the essential activator molecule of store-operated Ca2+ entry (SOCE) and a sorting regulator of certain ER proteins such as Stimulator of interferon genes (STING). Here, we characterize a conserved new variant, Stim1A, where splice-insertion translates into an additional C-terminal domain. We find prominent expression of exonA mRNA in testes, astrocytes, kidney and heart and confirm Stim1A protein in Western blot of testes. In situ, endogenous Stim1 with domain A, but not Stim1 without domain A localizes to unique adhesion junctions and to specialized membrane retrieval sites (tubulobulbar complexes) in testes. Functionally, using Ca2+ imaging and patch-clamp analysis, Stim1A shows a dominant-negative effect on SOCE and ICRAC, despite normal clustering and interaction with Orai1 investigated by combined TIRF and FRET analyses and as confirmed by an increased SOCE upon knock-down of endogenous Stim1A in astrocytes. Mutational analyses in conjunction with imaging and patch-clamp analyses of residues either in domain A or within the N-terminal region of Orai1 demonstrate a specific defect in stabilized channel gating. Our findings demonstrate that cell-type specific splicing of STIM1 adds both an intracellular targeting switch and adapts SOCE to meet the Ca2+ requirements of specific subcellular contact sites.

molecular biology↗

bioRxiv2020

ORAI1 channels form supra-molecular clusters at rest and agglomerate into distinct plasma membrane areas of oval and strand-like shapes after activation

The Ca2+ selective channel ORAI1 and endoplasmic reticulum (ER)-resident STIM proteins form the core of the channel complex mediating store operated Ca2+ entry (SOCE). Using liquid phase electron microscopy (LPEM) the distribution of ORAI1 proteins was examined at rest and after SOCE-activation at nanoscale resolution. The analysis of over seven hundred thousand of ORAI1 positions showed that already at rest, a majority of the ORAI1 channels formed STIM-independent distinct supra-molecular clusters. Upon SOCE activation and in the presence of STIM proteins, ORAI1 assembled in micron-sized two-dimensional (2D) structures, such as the known punctae at the ER plasma membrane contact zones, but also in divergent structures such as strands, and ring-like shapes. Our results thus question the hypothesis that stochastically migrating single ORAI1 channels are trapped at regions containing activated STIM, and we propose instead that supra-molecular ORAI1 clusters fulfill an amplifying function for creating dense ORAI1 accumulations upon SOCE-activation. STATEMENT OF SIGNIFICANCEORAI1 proteins form channels mediating store operated Ca2+ entry, an important trigger for many cellular functions, especially in the immune system. ORAI1 channels at rest are assumed to be randomly distributed in the plasma membrane, while they accumulate into so-called "punctae" upon activation, where binding by STIM proteins activate the Ca2+ channels. Using liquid phase electron microscopy, we discovered that ORAI1 forms small, elongated clusters indicating the existence of supramolecular assemblies. The role of such supramolecular organization of ORAI1 is possibly an amplifying function for the effective creation of ORAI1 accumulations in punctae, since the binding of only one ORAI1 protein would trap a multiple of channels.

biophysics↗